Bacteria are infamous for their fast replication rates, which are in part possible because of robust cell cycle mechanisms that ensure that virtually every division produces progeny with a full complement of the genetic material. In this project, we seek to understand the mechanisms involved in DNA partitioning and cell cycle control with the long-term goal of generating new strategies to control bacterial growth as bacteria remain a serious threat to human health. Our bacterial model of choice is the genetically tractable bacterium Caulobacter crescentus, a well-established cell cycle model for which synchronized cell cycle populations are easily obtained and whose cell cycle is conveniently associated with morphological transitions. In C. crescentus, chromosome segregation is initiated by the motion of the parS DNA sequence near the origin of replication. This active motion is dependent on the broadly conserved proteins ParA and ParB. Our first aim is to build on our previous findings to dissect the mechanism underlying the ParABS-dependent segregation mechanism. For this, we will use a battery of in vitro biochemical assays to examine the dimerization and ATPase activity of wild-type and mutant ParA proteins in the presence of varying concentrations of DNA, ParB and nucleotides. In addition, we will use super-resolution microscopy techniques to image the process of DNA segregation inside cells. Since the cell polarization factors TipN and PopZ affect the ParABS system, our second aim is to elucidate the function of these two proteins. Our recent findings suggest that TipN affects the directionality and speed of DNA segregation by sequestering ParA monomers. We will test this model using different ParA mutants and a combination of established biochemical, cytological and genetic assays. PopZ is known to assemble into a high-order structure at the cell poles where it tethers the ParB/parS partition complex and recruits cell cycle signaling proteins. Using a mutagenesis approach, we will address how PopZ achieves these activities and whether one activity (e.g., assembly into a high-order structure) is required for another (e.g., polar localization and/or interaction with ParB/parS). Our third aim is to address the intrinsic cell cycle mechanisms that regulate cell size in C. crescentus. This directly relates to the first two aims as previous work from our lab shows that the segregation of the ParB/parS complex is regulated by cell size and not time. Because of the coupling between DNA segregation, growth and division, defects in TipN, PopZ or the ParABS system result in cell size aberrations. We propose to perform a comprehensive single-cell study of various strains with different cell size distributions using the powerful image analysis software MicrobeTracker that we developed. This study will examine the contribution of each cell cycle event in cell size compensation, and provide new insight into cell cycle control.